Axonal transport plays a critical role in maintaining cellular and synaptic homeostasis that is essential for neuron development, function and survival. Impaired axonal transport is implicated in major neurological disorders. Our primary goal is to elucidate mechanisms regulating axonal transport of various membrane organelles. By employing live imaging of adult neurons from genetic mouse models combined with gene rescue experiments, we have made important discoveries as highlighted: (1) We revealed that snapin acts as an adaptor attaching dynein motors to endo-lysosomes and thus drives their retrograde transport from distal axons to the soma (Cai et al., Neuron 2010). Such a mechanism enables neurons to maintain effective degradation capacity and cellular homeostasis. (2) We revealed a motor-adaptor sharing model by which late endosome-loaded dynein-snapin transport machinery mediates the retrograde transport of autophagosomes upon their fusion into hybrid organelles named amphisomes, thus maintaining effective autophagic clearance in distal axons (Cheng et al., JCB 2015). (3) We showed that the endo-lysosomal pathway exerts a bipartite regulation of presynaptic activity. Late endosomal transport influences synaptic vesicle pool size by shuttling their components for degradation. Endosomal sorting determines synaptic vesicle positional priming (Di Giovanni et al., EMBO J, 2015). (4) We expanded our investigations using a familial Amyotrophic Lateral Sclerosis (fALS)-linked mouse model to provide in vitro and in vivo evidence that progressive lysosomal deficits are early fALS-linked pathological events in motor neurons (MNs), thus causing them to be more vulnerable to dying-back degeneration (Xie et al., Neuron 2015). Altogether, these studies advance our understanding of axonal endo-lysosomal trafficking that affects the maintenance of axonal and synaptic homeostasis, thus contributing to neurodegenerative diseases associated with dysfunction of synapses and autophagy-lysosomal system. These studies also build the solid foundation for the research accomplishments we have made in 2018-2019 fiscal year. Accomplishment 1: Provide Guidelines for Labeling Degradative Lysosomes in Nervous Systems (Cheng et al., Journal of Cell Biology 2018) Despite widespread distribution of lysosome-associated membrane proteins LAMP1/2 in lysosomes, endosomes, and the plasma membrane, LAMP1/2 have been routinely used markers for degradative lysosomes. This practice leads to misinterpretation of the roles of active lysosomes in neuronal growth, function, and survival in the healthy brain, and their pathological impact on neurodegenerative diseases. Thus, there is an urgent need to characterize LAMP1-labeled degradative and non-degradative organelles in nervous systems. We aimed to address two questions: (1) whether LAMP1-labeled organelles contain active forms of major lysosomal hydrolases and function as degradative lysosomes; and (2) whether altered LAMP1 distribution and intensity in diseased neurons represents a sensitive indicator for lysosomal deficits or lysosomal response to disease conditions. By applying immuno-electron microscopy, live degradative capacity analysis, and confocal and super-resolution imaging, we provided a quantitative analysis of LAMP1 distribution in various autophagic and endo-lysosomal organelles in neurons. Our study indicates that LAMP1/2 are neither specific markers to represent degradative lysosome distribution in neurons, nor a sensitive indicator to reveal the pathological response of lysosomes to disease conditions in vivo. We suggest that labeling a set of active lysosomal hydrolases would be more accurate than simply relying on LAMP1/2 staining. Therefore, our study provides guidelines for correctly labeling degradative lysosomes in in vitro and in vivo nervous systems, thus advancing our understanding of how lysosomal distribution, trafficking, and functionality contribute to neuronal health and disease progression. Accomplishment 2: Reveal the Mechanism Maintaining Axonal Degradation Capacity (Farfel-Becker et al., Cell Reports 2019) A long-standing question is whether axons are active degradation compartments with the capacity to locally eliminate protein aggregates and autophagosomes. The current accepted model is that mature lysosomes are mainly distributed in the soma, so that cargos destined for degradation need to be retrogradely transported into the soma for degradation. Interestingly, genetic or pharmacologic disruption of lysosomal function leads to axonal dystrophy, characterized by swellings containing accumulated degradation cargos. Lysosomal dysfunction, autophagy stress, and axonal dystrophy all contribute to the pathogenesis of major neurodegenerative diseases including Alzheimers, Parkinsons, and ALS. These clinical implications prompted us to address whether (1) neurons are able to recruit enzymatically active degradative lysosomes from the soma into distal axons to maintain local degradation capacity; and (2) these axon-targeted lysosomes are locally degrading autophagosomes and aggregate-prone mutant proteins. We characterized the axonal delivery of degradative lysosomes by applying a set of fluorescent probes that selectively label active forms of lysosomal hydrolases on cortical neurons cultured in microfluidic devices. We demonstrated that degradative lysosomes are dynamically delivered to distal axons in developing and mature neurons from both central and peripheral nervous systems; additionally, disrupting the axonal delivery induces autophagic stress with build-up of autophagosomes and Parkinson disease-related mutant -synuclein cargos. Thus, axonal degradation capacity is maintained by delivery of fresh degradative lysosomes from the lysosomal reservoir in the soma. Our study establishes a foundation for investigations into axonal lysosome trafficking and functionality in neurodegenerative diseases and LSDs associated with axonal pathology and autophagy stress. Accomplishment 3: Elucidate Axonal Transport Mechanism underlying Autism-like Synaptic and Behavioral Abnormalities (Xiong et al., manuscript under invited revision) The formation and maintenance of synapses require long-distance axonal delivery of newly synthesized synaptic proteins from the soma to distal synapses, raising the fundamental question of whether impaired axonal transport is associated with the neurodevelopmental disorder autism. Our previous studies revealed that syntabulin (STB) is a kinesin-1 motor adaptor that links the motor to cargos containing Bassoon and multiple presynaptic components, thus contributing to presynaptic assembly and maintanence. The stb gene locates within the autism susceptibility loci 8q22-24. A recent whole exome sequencing study identified an autism-linked de-novo STB missense variant. Thus, there is an urgent need to establish a mechanistic link between impaired transport of presynaptic cargoes and autism-like synaptic and social behavioral abnormalities. Generating a conditional stb knockout (stb cKO) mouse provides us an in vivo model system to address this fundamental question. We report that stb cKO mouse brains display impaired axonal transport of presynaptic cargos, reduced formation and maturation of synapses, and altered synaptic transmission. Intriguingly, stb cKO mice exhibit core autism-like traits, including defective social recognition and communication, increased stereotypic behavior, and impaired spatial learning and memory. Our study was confirmed by an autism-associated STB missense variant that loses its motor adaptor function and fails to rescue stb cKO phenotypes. Therefore, our study reveals that altered axonal transport is one of core presynaptic mechanisms underlying synaptic dysfunction and social behavioral abnormalities that bear similarities to autism.